a. Experimental design
A cross-sectional experimental study was carried out in the operation room of Hospital Santa Rita no complexo Santa Casa de Porto Alegre, from August 15, 2019, to April 15, 2020 with a total of 31 aerosolizations. PIPAC applications were simulated in a negative pressure operating room with unidirectional (laminar) airflow ventilation and open sealing doors to analyze contamination in the operating room corridor. The first stage of validation of the methodology under analysis, 10 aerosolization tests were carried out simulating different levels of leakage to assess the contamination scenarios. The first scenario was the simulation of leakage keeping the trocar’s luer lock open throughout the procedure. In this scenario, the contamination was hard to measure. The second scenario was the aerosolization 20 cm above the center of the operating table in a 10-liter open container. The third model was free aerosolization 20 cm above the center of the operating table (Figure-1). After assessing the first 10 applications, the scenario that proved to be most suitable for the purpose was scenario 3 During all procedures, no contamination control method was used. This study was submitted to the Ethics Committee, however, it was exempt from analysis because it is an experimental study that does not involve living beings or toxic substances.
b. Aerosolization procedure
The following equipment was used: BhioQAP registered at the Agência Nacional de Vigilância Sanitária (ANVISA) under No. 80381210072 and IV contrast injection system with remote actuation (Empower CTA-Bracco). The aerosolized equipment was kept in the center and 20 cm above the operating with the aid of a mechanical arm that supported the system (Figure-1).
The assessment of environmental contamination during the PIPAC procedure was performed using a 1% aqueous solution of caffeine (Sigma-Aldrich®). This substance was specifically chosen because caffeine has low toxicity and is easy to detect. Some physical and chemical characteristics of caffeine are described in table 1.
The injector (Empower CTA-Bracco) was configured to apply the fixed volume for each 200 ml application. The injection equipment was always programmed with fixed infusion parameters of 3.0 ml/s with a maximum pressure of 200 to 300 PSI. The injection equipment adjusts the infusion flow to keep the pressure below 300 PSI. The average injection variation was 0.6 ml/s ± 0.2 ml/s. All injections were carried out without locking the injection equipment or changing the parameters described.
Cellulose nitrate membranes (8.00 µm pores and 47 mm diameter/ UNIFIL®) were used to capture the caffeine concentration in different parts of the operating room for 5 fixed periods of exposure. The membrane exposure times after the start of aerosolization were 2, 5 15 and 30 minutes. The 5th exposure period of the cellulose membrane was started 30 minutes after the beginning of the aerosolization up to 35 minutes. A escolha do tempo foi relacionada a períodos críticos do processo de aerossolização. The time was chosen based on critical periods in the aerosolization process. Time 2 is related on average to half the aerosolization time. Time 5 regards the end of aerosolization in the vast majority of applications. Time 15 relates to half the PIPAC procedure time in clinical practice. Time 30 is related to the end of the proposed procedure in clinical practice. The time between 30-35 relates to the end of PIPAC procedure and entry of the assistant team in the surgical environment. The areas of interest for determining the contamination were established at 6 points of interest. The determination of these points was chosen according to the occupational risk of healthcare professionals or by the places of a high risk of environmental contamination. (Figure-2) The sites chosen for data collection, representing the following positions during PIPAC procedure: (1) patient, (2) surgeon site, (3) anesthetist site, (4) below the injection site near the injector, (5) at the airflow outlet (which remained turned off during the procedure) and (6) under the operating room door frame.
c. Environment contamination analysis
Cellulose nitrate membranes collected in each situation were analyzed to determine the environmental concentration of caffeine. After the collection period in each different scenario, the membranes were separately packed in plastic bags and sent to Central Analítica da Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA). Immediately after arriving at the assessment site, the samples were placed in an amber flask containing 3 ml of water supplemented with 0.1% formic acid, protected from light and incubated at 4-8 ºC for 24 hours. Aliquots of the solutions were directly injected into a liquid chromatography system together with high-resolution mass spectrometry (LC-HRMS). This system was integrated into a mass spectrometer composed of a hybrid system with quadrupole analyzer and TOF (time of flight, Bruker Daltonics, micrOTOF-QIII model) in series and in an orthogonal position for high resolution and mass accuracy. Data were processed using Data Analysis e HyStar™ software. The analyses were carried out through electrospray ionization (ESI) and the parameters such as ionization method, temperature, gas flow, collision energy and capillary energy were tested and optimized. Elution was made on a Shim-pack XR-ODS II chromatography column (75 x 2 mm, particle size 2.2 µm, Shimadzu®, Tokyo, Japan) with a water and methanol gradient supplemented with 0.1% formic acid, 0.4 ml/min flow, and temperature of 50 ºC. The total assessment time was on average 4 minutes.
d. Statistical analysis
The sample size was calculated using the WINPEPI program (Abramson, J.H. WINPEPI updated: computer programs for epidemiologists, and their teaching potential. Epidemiologic Perspectives & Innovations 2011, 8:1) considering a 5% significance level and 90% power. The first 10 aerosolizations were used to validate the experimental design and optimize the analytical methodology, define better packaging, train the research team, and choose the best-proposed scenario. After the tenth aerosolization, 21 consecutive aerosolizations were considered for analysis. The variable distribution was assessed by the Kolmogorov-Smirnov test. Differences in the intervals of 0 to 2 minutes, 2 to 5 minutes, 5 to 15 minutes, and 15 to 30 minutes were calculated, and this difference was divided by 2, 3, 10 and 15 minutes to determine the variation per minute. The variations per minute were described by the median and the 25-75% interquartile range and compared using the Friedman test between the intervals. A 5% significance level was considered.